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MiceMacrophage Defects in Autoimmune BXD2of Apoptotic Antigens Due to Marginal Zone Cutting Edge: Defective Follicular Exclusion

Hui-Chen Hsu and John D. MountzHao Li, Qi Wu, Jun Li, PingAr Yang, Zilu Zhu, Bao Luo,

ol.1300041http://www.jimmunol.org/content/early/2013/03/28/jimmun

published online 29 March 2013J Immunol 

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Cutting Edge: Defective Follicular Exclusion of ApoptoticAntigens Due to Marginal Zone Macrophage Defects inAutoimmune BXD2 MiceHao Li,*,† Qi Wu,* Jun Li,* PingAr Yang,* Zilu Zhu,‡ Bao Luo,* Hui-Chen Hsu,* andJohn D. Mountz*,†,x

Marginal zone macrophages (MZMs) act as a barrier toentry of circulating apoptotic debris into the folliclesof secondary lymphoid organs. In autoimmune BXD2mice, there is a progressive reduction in the functionand numbers of MZMs. Absence of MZMs results inretention of apoptotic cell (AC) debris within the mar-ginal zone (MZ) and increased loading of AC Ags onMZ B cells and MZ-precursor (MZ-P) B cells. TheMZ-P B cells are capable of translocating the AC Agsto the follicular zone and stimulating T cells. BothMZMs and MZ-P B cells from BXD2 mice expresslow levels of tolerogenic signals and high levels of in-flammatory signals. Thus, the current study suggests amultifaceted mechanism in which MZMs maintain tol-erance to apoptotic autoantigens and suppress their trans-location to follicles. Lack of clearance of apoptotic debrisby MZMs drives follicular Ag–transportation by MZ-PB cells to stimulate an autoimmune response. TheJournal of Immunology, 2013, 190: 000–000.

Apoptotic cells (ACs) contain “blebs” that are highlyenriched with autoantigens, including nucleosomalDNA and small nuclear riboproteins (1, 2). Nor-

mally, the corpses of the ACs are removed locally by tissuemacrophages (3). ACs and debris that escape this clearancemechanism are prevented from entering the follicles of sec-ondary lymphoid organs by the marginal zone (MZ). There isconsiderable evidence that loss of integrity of the MZ barrierthat results in the lack of clearance of AC autoantigens cancontribute to development of autoimmune disease (4–8). TheMZ macrophages (MZMs), which express the scavenger re-ceptor SR-A and MARCO to sense and remove waste mate-rials (8, 9), are a critical component of this barrier. In mice,autoimmune responses can be induced or promoted by either

targeted deletion of SH2 domain–containing inositol 59-phosphatase 1 (SHIP-1) in myeloid cells (9), which results indislocations of MZMs, or treatment with an appropriate doseof clodronate liposome (CL), which results in loss of MZMs(5, 6).BXD2 mice develop a lupus-like autoimmune disease (10).

The generation of the high-affinity pathogenic autoantibodiesis associated with the development of large spontaneous ger-minal centers (GCs) in the spleen (11–13). However, themechanisms that permit the initial exposure of the B cells tothe autoantigens have not been elucidated. The production ofautoantibodies in BXD2 mice with specificity for Ags derivedfrom a wide range of cellular compartments, including fila-ment proteins, chromatin, and ribonuclear components (14),is consistent with the concept that autoantigens can be derivedby proteolytic cleavage during the process of apoptosis (1).The present results confirm that defective clearance of apo-ptotic debris by MZMs and the associated transport of ACAgs by B cells can provoke a T cell response in BXD2 mice.

Materials and MethodsMice

Wild-type C57BL/6 (B6), C57BL/6-Tg(TcraTcrb)425Cbn/J (OT-II TCRtransgenic [Tg]), B6-Rag22/2, C57BL/6-Tg(CAG-OVA)916Jen/J, transgenicmice expressing the membrane-bound chicken OVA on all cell surfaces(mOVA Tg), and BXD2 recombinant inbred mice were obtained from TheJackson Laboratory (Bar Harbor, ME). B6-Rag22/2 or B6-OT-II TCR Tgmice were backcrossed with BXD2 mice for .10 generations to generateBXD2 OT-II TCR Tg or BXD2-Rag22/2 mice, which were further inter-crossed to generate BXD2 OT-II TCR Tg Rag22/2 mice.

Apoptotic cells

For in vivo administration of ACs, mice were injected i.v. with 2 3 107 ap-optotic thymocytes. For depletion of MZMs, mice were treated i.v. with150 mg CL (Encapsula NanoSciences, Nashville, TN) 4 h before AC transfer,as described (5). Control mice were treated with PBS liposomes. The dose forselective delivery to MZMs was optimized using Fluoroliposome (EncapsulaNanoSciences).

*Division of Clinical Immunology and Rheumatology, Department of Medicine, Uni-versity of Alabama at Birmingham, Birmingham, AL 35294; †Department of Microbi-ology, University of Alabama at Birmingham, Birmingham, AL 35294; ‡Division ofHematology and Oncology, Department of Medicine, University of Alabama at Bir-mingham, Birmingham, AL 35294; and xBirmingham VA Medical Center, Birming-ham, AL 35294

Received for publication January 17, 2013. Accepted for publication March 1, 2013.

This work was supported by a VA Merit Review Grant (1I01BX000600-01), as well asgrants from the National Institutes of Health/National Institute of Allergy and InfectiousDisease (1AI 071110, ARRA 3RO1AI71110-02S1, and 1RO1 AI083705), The Amer-ican College of Rheumatology Research and Education Foundation, Arthritis Founda-tion, and Lupus Research Institute. Flow cytometry and confocal imaging were carriedout at the University of Alabama at Birmingham Comprehensive Flow Cytometry Core

(P30 AR048311 and P30 AI027767) and University of Alabama at Birmingham Ana-lytic Imaging and Immunoreagents Core (P30 AR048311), respectively.

Address correspondence and reprint requests to Dr. Hui-Chen Hsu and Dr. John D.Mountz, Division of Clinical Immunology and Rheumatology, Department of Medi-cine, University of Alabama at Birmingham, Room 307, Shelby Building, 1825 Uni-versity Boulevard, Birmingham, AL 35294-0007. E-mail addresses: rheu078@uab.edu(H.-C.H.) and jdmountz@uab.edu (J.D.M.)

The online version of this article contains supplemental material.

Abbreviations used in this article: AC, apoptotic cell; CL, clodronate liposome; FO,follicular; GC, germinal center; mOVA, membrane-bound chicken OVA; MZ, marginalzone; MZM, marginal zone macrophage; MZ-P, marginal zone precursor; RPM, red pulpmacrophage; SHIP-1, SH2 domain–containing inositol 59-phosphatase 1; Tg, transgenic.

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CD4 T cell stimulation

Splenic CD4 cells from BXD2 OT-II TCR Tg Rag22/2 mice were labeledwith CFSE (5 mM) and transferred i.v. into recipient mice (2 3 106/mouse).Recipient mice were administered apoptotic thymocytes prepared frommOVA Tg mice 12 h later and sacrificed after an additional 72 h.

In some experiments, red pulp macrophages (RPMs) (F4/80+CD11blo) orfollicular (FO; CD19+IgMloCD21loCD23hi), MZ (CD19+IgMhiCD21hi

CD23lo), or MZ-precursor (MZ-P; CD19+IgMhiCD21hiCD23hi) B cellswere FACS sorted from mice that were injected with mOVA+ ACs 30 minprior to sacrifice. In other experiments, sorted RPMs or FO, MZ, or MZ-PB cells (53 105 each) from naive 2-mo-old BXD2 mice were cocultured withpurified and CFSE (1 mM)–labeled OT-II CD4 T cells (2 3 105) in thepresence or absence of either OVA protein or OVA323–339 peptide, asdescribed previously (15). In all in vitro experiments, CD4 T cell–prolifer-ative response was determined by the reduction in CFSE intensity by flowcytometry 3 d after coculture.

Reagents and analyses

Flow cytometry and immunofluorescence microscopy were carried out aspreviously described (12). Real-time quantitative PCR was carried out, asdescribed (12), using the following primers: Il6, 59-TCCAGTTTGGTAG-CATCCATC-39 (forward), 59-CCGGAGAGGAGACTTCACAG-39 (reverse);Il10, 59-AATTCCCTGGGTGAGAAG-39 (forward), 59-TGCAGTTGATG-ATGAAGATGTC-39 (reverse); and Tgfb: 59-CTACTATGCTAAAGAG-GTCAC-39 (forward), 59-CATGTTGCTCCACACTTG-39 (reverse). ForWestern blot analysis, anti-Btk (Santa Cruz), anti-IDO (Abcam), anti-GAPDH,anti-SHIP1, and anti-rabbit HRP-conjugated Ab (Cell Signaling Technology)were used.

Results and DiscussionReduction of MZMs in BXD2 mice

Confocal imaging (Fig. 1A) and FACS analysis (Fig. 1B) wereused to determine the integrity and enumerate the percentageof SIGN-R1+ MZMs in the spleens of autoimmune BXD2mice. There was an age-dependent loss of MZMs in theBXD2 mice but not in the B6 mice (Fig. 1A, 1B). The totalnumbers of F4/80negCD11bloI-AbnegSIGN-R1+ MZMs (16)were equivalent in 3-mo-old B6 and BXD2 mice, but therewas a dramatic loss of these cells in 9-mo-old BXD2 mice butnot B6 mice (Fig. 1B). There was an age-related defect in theclearance of ACs, as indicated by an increase in the numberof TUNEL+ ACs found near the MZMs and inside areas ofthe follicle distinct from the GCs in BXD2 mice (Fig. 1C).Consistent with these, there were age-related increases in thelevels of circulating autoantibodies for MARCO, SR-A, andDNA in BXD2 mice (Fig. 1D). Elevation of IgG anti-MARCO and anti–SR-A was found in the serum of lupuspatients and mouse models of lupus; these autoantibodiespotentially affect macrophage phagocytosis of ACs (7).

Capture of AC debris by B cells and their intrafollicular migration

The functional activities of the MZMs were evaluated by theability to clear exogenous CFSE-labeled apoptotic thymocytes(Fig. 2A). Compared with B6 mice, the apoptotic thymocytespersisted for an extended period of time in 2-mo-old BXD2mice. Thus, the functional defects in the MZMs precede theloss of these cells and represent one of the earliest autoim-mune defects detected in the BXD2 strain.However, the failure to clear Ags alone is not sufficient to

induce an autoimmune response in BXD2 mice, because largeGCs are found inside the follicles. Cyster and colleagues (15)showed that B cells can shuttle between the MZ and FO areas,thereby facilitating Ag transport. We established previouslythat, in BXD2 mice, there is the expansion in number of MZ-PB cells that is associated with the reduction in the MZ B cellsand the heightened Ag delivery by MZ-P B cells (17, 18).

To determine whether BXD2 B cells can capture and deliverAC Ags in MZM-deficient conditions, we tracked CFSE-labeled apoptotic thymocytes in mice that were treated with150 mg of CL. This dose was used because it selectively in-duced uptake of liposomes by MZMs (Supplemental Fig.1A). In mice administered CL, there was a time-dependentcapture of ACs by MZ B and MZ-P B cells but not FO B cells(Fig. 2B, Supplemental Fig. 1B). The numbers of CFSE+

cells, which were located predominantly in the MZ, peaked at5–15 min, and the majority was processed at 60 min (Fig. 2C,left panel ). This may be associated with the higher uptake ofCFSE by MZ B cells compared with MZ-P B cells duringthese early time points (Fig. 2B, Supplemental Fig. 1B).Within 5–30 min, there was a rapid migration of MZ-PB cells to the periphery of the follicle and into the MZ (Fig.2C, right panel, Supplemental Fig. 1C, 1D). By 1 h after

FIGURE 1. Age-related decrease in MZMs in BXD2 spleens. (A–C) Naive

B6 and BXD2 mice were sacrificed at the indicated age. (A) Confocal imaging

showing the decline of MZMs in the spleen in BXD2 mice. Staining was

carried out using anti–SIGN-R1 (eBioscience). Original magnification 350.

(B) FACS analysis showing the frequency of CD11bintF4/80loSIGN-R1+I-

Abneg MZMs (rectangle gated cells) in the spleen of B6 and BXD2 mice (leftand middle panels). MZM cell count was calculated by multiplying the per-

centage of MZMs by the total spleen cell count obtained from each mouse

(right panel ). (C) Confocal imaging showing the anatomic location of ACs

(TUNEL+) relative to the location of MZMs (MARCO+) and GCs (PNA+) in

the representative follicle (left panel ). Staining was carried out using an anti-

MARCO Ab (AbD Serotec), peanut agglutinin (PNA, Vector Laboratories) and

an In Situ Apoptosis Detection Kit (EMD Millipore). High-power view of the

boxed areas (right panel ). Original magnification 3200. (D) ELISA analysis of

serum titers of the indicated autoantibodies in the indicated strains. All data are

mean 6 SEM (n = 3–4/group) for two independent experiments. *p , 0.05,

**p , 0.01, ***p , 0.005, B6 mice versus BXD2 mice of the same age.

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administration of the ACs, the majority of MZ-P B cells, butnot MZ B cells, exhibited massive inward migration into thefollicles. At this time point, the majority of both the MZ-Pand MZ B cells carried the apoptotic debris (Fig. 2B, 2C,Supplemental Fig. 1B).

Apoptotic Ags capturing MZ-P B cells stimulate CD4+ T cells

To determine whether MZMs are mechanistically required tosuppress the T cell response to ACs and associated Ags, wetransferred CFSE-labeled OT-II TCR Tg T cells into BXD2and B6 mice and assessed the response of Vb5+ T cells toapoptotic thymocytes derived from mOVA Tg mice. Signif-icantly higher numbers of OT-II T cells entered the cell cycle

in the spleens of the 2- and 9-mo-old BXD2 mice than thespleens of age-matched B6 mice postapoptotic mOVA+ thy-mocyte transfer (Fig. 3A, right panel ). The percentages ofCFSE-labeled OT-II T cells that entered the cell cycle in miceadministered CL were similar to that observed in 9-mo-oldBXD2 mice (Fig. 3A, right panel ), which is consistent withthe dramatic loss of MZMs observed in the older BXD2 mice(Fig. 1A, 1B).We showed previously that the T cell costimulatory function

of MZ-P B cells is superior to other B cells in BXD2 mice (17,18). Therefore, we cocultured CFSE-labeled OT-II CD4+

T cells with F4/80+ RPMs or subpopulations of B cells sorted

FIGURE 2. Defective clearance of apoptotic debris is associated with in-

creased uptake of ACs by MZ/FO shuttling MZ-P B cells in the spleens of

BXD2 mice. (A) Confocal imaging showing the kinetics of clearance of ad-

ministered CFSE-labeled apoptotic thymocytes (pseudocolor red) in the MZ

area in the spleen of 2-mo-old mice (left panel ). High-power view of the

indicated (box) areas (right panel ). Original magnification 3200. (B and C)

BXD2 mice (2 mo) were treated with either CL- or PBS-liposome 4 h prior to

CFSE-labeled apoptotic thymocyte administration. Following AC adminis-

tration, mice were sacrificed at the indicated time points. (B) FACS analysis of

subpopulations of B cells that were positive for AC Ags (CFSE+) (left panel ).FO, MZ, and MZ-P B cells were gated as described (17, 18). Confocal

imaging assessment of the binding of FACS-sorted MZ or MZ-P B cells with

CFSE+ (pseudocolor red) apoptotic debris (right panel ). A representative cell

from the indicated subset is shown. Original magnification 31000. (C)

Confocal imaging showing a representative spleen follicle from each group to

reveal the clearance of the administered CFSE+ apoptotic thymocytes (pseu-

docolor red) in the spleen (left panel ) and the anatomic location of FO (red:

CD1d2CD23+), MZ (green: CD1d+CD232), MZ-P (yellow: CD1d+CD23+),

and GC (blue: PNA+) in each group (right panel ). Original magnification

3200. (n = 2–3 mice/group for three independent experiments).

FIGURE 3. Stimulation of CD4 T cells by MZ-P B cells carrying AC Ags.

(A) Flow cytometry analysis of the percentage of the transferred BXD2 OT-II

CD4 T cells (TCR Vb5) in total spleen cells in the indicated recipient groups

(left panels). Flow cytometry analysis of the in vivo OVA Ag-specific prolif-

erative response of the transferred OT-II TCR CD4 T cells, indicated by

attenuation of CFSE intensity (right panels). Control mice (2 mo old) were

administered CL. (B) Bar graph represents the in vitro proliferative response

of OT-II TCR CD4 T cells (CFSElo) 72 h after coculture with the indicated

cell populations, which were FACS sorted from the spleens of mice 30 min

postadministration of apoptotic mOVA+ thymocytes. (C) Flow cytometry

analysis of the in vitro OT-II T cell–proliferative response (CFSElo) to either

OVA protein or the OVA323–339 peptide processed or presented by the in-

dicated populations of cells that were FACS sorted from naive 2-mo-old

BXD2 mice. CFSE-labeled OT-II T cells alone were used as control. (D) RT-

PCR analysis of the expression of the indicated gene in sorted MZMs and

MZ-P B cells derived from the spleen of naive mice. (E) Western blot analysis

of the expression of the indicated protein in sorted CD11b cells from naive

mice (left panel ). ImageJ analysis of the intensity of each protein after nor-

malization to the intensity of GAPDH (n = 3–4/group) (right panel ). *p ,0.05, **p , 0.01, ***p , 0.005 versus the same population of cells from B6

mice in (A), (B), (D), and (E) or from OVA protein–stimulated cells in (C).

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from the spleens of BXD2 mice that had been administeredmOVA thymocytes. These results show that both MZ andMZ-P B cells can present Ags derived from ACs, and this Agpresentation and stimulation of T cells are higher in BXD2mice compared with B6 mice (Fig. 3B, Supplemental Fig. 2).Although the ability of MZ-P B cells to process intact OVAproteins for stimulation of CD4+ T cells was lower than thatof RPMs, the ability of MZ-P B cells to present OVA peptidefor stimulation of T cells was superior to that of all othertested B cell populations from BXD2 spleens (Fig. 3C). Thissuggests that MZ-P B cells that have migrated into the FOregion may either directly process or capture processed ACAgs, thereby stimulating CD4+ T cells.Under normal conditions, phagocytic cells that ingest ACs

produce tolerogenic signals, including TGF-b and IL-10,which maintain tolerance to self-Ags (19). Both MZMs andMZ-P B cells from 3-mo-old BXD2 mice exhibited lowerexpression of genes encoding tolerogenic cytokines, includingTGF-b and IL-10, and higher expression of the gene encodingthe immunogenic cytokine IL-6 compared with those from B6MZMs and MZ-P B cells (Fig. 3D). IDO was recently shownto be an important tolerogenic signal inducer in MZMs of B6mice following AC administration (6). However, IDO wasdetectable in MZMs of unmanipulated younger MRL-Faslpr/lpr

mice, and its inhibition with D-1-methyl-tryptophan pro-moted autoimmunity (6), suggesting that low IDO can en-hance, but is not the primary cause of, autoimmunity in MRL-Faslpr/lpr mice. In contrast, to our knowledge, the current studydemonstrated for the first time that CD11b+ cells from amouse model of spontaneous autoimmunity expressed sig-nificantly lower levels of IDO compared with those fromnormal B6 mice (Fig. 3E). Also, local high levels of IL-6 in theMZ area might contribute to an immunogenic signal for ACAgs, because IL-6 degrades IDO (20). The TGF-b–IDOITIMs stimulate SHIP-1 (Inpp5d ), and the TGF-b–IDO–SHP axis was recently identified as the major long-term tol-erogenic signal in DCs (21). There also were dramatically lowerlevels of SHIP-1, together with higher levels of its counteractingmolecule, Btk (9), in the CD11b+ cells from the spleens ofBXD2 mice (Fig. 3E). These findings are consistent with theobservations that loss of the expression of SHIP-1 in myeloidcells of Lyz.Cre Ship12/2 mice was associated with disruptedMZM integrity and enhanced splenomegaly and that deficiencyof Btk in Lyz.Cre Ship12/2 mice reversed these phenotypes (9).In summary, we showed previously that the development of

the high titers of autoantibodies in BXD2 mice is dependenton abnormalities in two compartments in the spleen: enhancedproduction of type I IFNs in the MZ induces FO translocationof Ag-bearing MZ-P B cells, which costimulate T cells (17,18), and enhanced IL-17 production in the GCs that upre-gulates regulator of G-protein signaling proteins, therebyenhancing T–B cell interactions (12, 22). We now have ob-served that these abnormalities are preceded by the develop-ment of defects in the MZ barrier in BXD2 mice. Collectively,the present results suggest a novel mechanism of autoimmunityin which “leaks” in the FO exclusion of self-apoptotic Ags,together with enhanced activity of carrier B cells, subvertsotherwise protected adaptive immune processes in the follicles.McHeyzer-Williams et al. (23) elegantly described the impor-tance of B cells as initial Ag-transporting cells in adaptiveimmunity. The present study directly demonstrates that, in

a situation in which MZM apoptotic debris ingestion andtolerogenic functions are defective, FO translocating immu-nogenic B cells can capture and deliver the autoantigens intothe follicles, thus providing the previously unknown bridgingmechanism between defective clearance of apoptotic debrisand the development of autoantibodies. Strategies that restoreand retolerize MZM function may represent a novel approachto attenuation of autoimmune disease.

AcknowledgmentsWe thank Dr. Fiona Hunter for review of the manuscript and Karen Beeching

for excellent secretarial assistance.

DisclosuresThe authors have no financial conflicts of interest.

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